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Background: The efficacy and safety of thrombomodulin alfa (TM-α), a cofactor protein promoting thrombin-mediated protein C activation, have been examined in a phase 3 randomized, double-blinded, parallel-group trial in Japan. We have previously reported that TM-α is noninferior to heparin for the resolution of disseminated intravascular coagulation (DIC)., Objective: To investigate the basis for the efficacy of TM-α in the phase 3 clinical trial in Japan through post hoc analysis of coagulation and fibrinolysis parameters., Patients/methods: The 227 patients of the full analysis set population described in the original phase 3 trial in Japan were included in this analysis. Changes in parameters between before and after TM-α or heparin administration in each of the two patient groups, with underlying diseases of either hematologic malignancy or infection, were studied separately and results were compared between TM-α and heparin treatment groups in a post hoc manner., Results: TM-α administration did not prolong activated partial thromboplastin time but significantly decreased thrombin-antithrombin complex levels compared with heparin treatment. TM-α administration reduced consumption of endogenous anticoagulants such as antithrombin and protein C by DIC, compared with the heparin group. DIC scores were decreased in both TM-α and heparin groups during the 6-day treatment., Conclusion: TM-α can alleviate intravascular coagulation and consumption of anticoagulants without extending coagulation times. This may be associated with the relatively low risk of bleeding during TM-α treatment., (© 2020 The Authors. Research and Practice in Thrombosis and Haemostasis published by Wiley Periodicals LLC on behalf of International Society on Thrombosis and Haemostasis.)

Tumor cells secrete membrane vesicles of various sizes, termed extracellular vesicles (EVs), which have gained increasing attention as potential tumor diagnostic markers. Tumor-derived EVs are enriched with high-mannose-type glycans. Here, we report the affinity isolation of EVs from human melanoma A375 cells by using high-mannose-type glycan-specific agglutinin from Oscillatoria Agardhii (OAA). Glycan analysis of melanoma EVs revealed the presence of high-mannose-type glycans with structural units preferred by OAA. We showed that in solution, OAA binds to melanoma EVs in a high-mannose-type glycan-dependent manner. Furthermore, OAA-immobilized beads were found to capture 60% of the particles and most proteinous components from melanoma EVs. Major EV glycoproteins that potentially interact with OAA were identified to be cluster of differentiation 109 (CD109), integrin α6 and a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). In addition to melanoma EVs, OAA captured EVs from human lung cancer, glioblastoma and colon cancer cells, but not those from endothelial cells and fibroblasts. These results indicate that OAA-immobilized beads may serve as a novel platform for affinity-capture of tumor-derived EVs., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Cells secrete heterogeneous populations of extracellular vesicles (EVs) via unknown mechanisms. EV biogenesis has been postulated to involve lipid-protein clusters, also known as membrane microdomains., Methods: Membrane properties and heterogeneity of melanoma-derived EVs were analyzed by a detergent solubilization assay, sucrose density gradient ultracentrifugation and immunoprecipitation. EV secretion was modulated by RNA interference and pharmacological treatments., Results: We identified two EV membranes (low-density exosomal detergent-insoluble membranes [EV-DIMs]; EV detergent-soluble membranes [EV-DSMs]) and discovered an abundant, novel type of high-density EV-DIMs. The high-density EV-DIMs accumulated the microdomain-resident protein flotillin-1, as well as a disintegrin and metalloproteinase domain containing protein 10 (Adam10), the hepatocyte growth factor receptor Met and its proteolytic fragments. Low-density EV-DIMs also contained flotillin-1. EV-DSMs were enriched with tetraspanin CD81, melanogenic enzymes and proteolytic fragments of Adam10. Intact and fragmented forms of Adam10, which resided in distinct membrane types, were secreted by different EVs. The fragmented form of Met was associated with DIMs much more efficiently than the intact form and they were secreted by distinct EVs. We identified that the endosomal sorting complexes required for transport machinery was indispensable for EV secretion of both mature and fragmented forms of Adam10 and Met., Conclusion: The findings of this study reveal the role of the interplay between membrane organization and sorting machineries in generating the heterogeneity of EVs., General Significance: This study provides novel insights into important aspects of EV biogenesis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Disseminated intravascular coagulation (DIC) is an acquired thrombo-haemorrhagic disorder which arises in clinical scenarios like sepsis, trauma and malignancies. The clinic-laboratory diagnosis of DIC is made in a patient who develops the combination of laboratory abnormalities in the appropriate clinical scenario. The most common laboratory parameters in this setting have been the clotting profile, platelet count, serum fibrinogen and fibrin degradation markers. These tests had the advantage that they could be performed easily and in most laboratories. However, with the better understanding of the pathophysiology of DIC, in recent years, more specific tests have been suggested to be useful in this setting. The newer tests can also prove to be useful in prognostication in DIC. In addition, they may provide assistance in the selection and monitoring of patients diagnosed with DIC., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Secondhand cigarette smoke exposure (SSE) has been linked to carcinogenic, oxidative, and inflammatory reactions. Herein, we investigated whether premature skin aging could be induced by SSE in a rat model, and assessed the cytoplasmic translocation of high mobility group box 1 (HMGB1) protein and collagen loss in skin tissues. Animals were divided into two groups: SSE and controls. Whole body SSE was carried out for 12 weeks. Dorsal skin tissue specimens were harvested for HMGB1 and Mallory's azan staining. Correlations between serum HMGB1 and collagen levels were determined. Rat skin exposed to secondhand smoke lost collagen bundles in the papillary dermis and collagen decreased significantly (p<0.05) compared with control rats. In epidermal keratinocytes, cytoplasmic HMGB1 staining was more diffuse and there were more HMGB1-positive cells after four weeks in SSE compared to control rats. A negative correlation between HMGB1 serum and collagen levels (r=-0.631, p=0.28) was also observed. Therefore, cytoplasmic HMGB1 expression in skin tissues might be associated with skin collagen loss upon the initiation of SSE. Additionally, long-term SSE might affect the appearance of the skin, or could accelerate the skin aging process., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Chronic low-grade inflammation is a key contributor to high-fat diet (HFD)-related diseases, such as type 2 diabetes, non-alcoholic steatohepatitis, and atherosclerosis. The inflammation is characterized by infiltration of inflammatory cells, particularly macrophages, into obese adipose tissue. However, the molecular mechanisms by which a HFD induces low-grade inflammation are poorly understood. Here, we show that histone H3, a major protein component of chromatin, is released into the extracellular space when mice are fed a HFD or macrophages are stimulated with the saturated fatty acid palmitate. In a murine macrophage cell line, RAW 264.7, palmitate activated reactive oxygen species (ROS) production and JNK signaling. Inhibitors of these pathways dampened palmitate-induced histone H3 release, suggesting that the extracellular release of histone H3 was mediated, in part, through ROS and JNK signaling. Extracellular histone activated endothelial cells to express the adhesion molecules ICAM-1 and VCAM-1 and the procoagulant molecule tissue factor, which are known to contribute to inflammatory cell recruitment and thrombosis. These results suggest the possible contribution of extracellular histone to the pathogenesis of HFD-induced inflammation and thrombosis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Obesity-induced glucose and lipid metabolism disorders have become risk factors for lifestyle diseases. Powderized Parachlorella beijerinckii (BP) and its hot water extract (BCEx) are believed to be useful for preventing common diseases such as hypertension, arteriosclerosis, and hyperlipidemia. The present study investigated how chlorella components influence common diseases in obese mice and rats on a high-fat diet., Methods: We fed C57BL/6J mice a high-fat diet containing 5% BP, and then weighed their organs, tested their glucose tolerance and insulin sensitivity, and analyzed their serum. Further, we fed Sprague-Dawley rats with a high-fat diet containing 1% BCEx, and then weighed their organs and analyzed their serum parameters., Results: BP administration had no effect on high-fat diet-induced obesity. However, compared with high-fat diet group, BP group had improved glucose tolerance and insulin sensitivity and inhibited the hypertrophic growth of visceral fat cells. In addition, BP group had improved serum adiponectin, leptin and monocyte chemoattractant protein-1 (MCP-1) levels. The MCP-1 expression level at epididymal fat was decreased at BP group. BCEx administration reduced amount of peritesticular fat and serum triglyceride (TG) levels., Conclusions: The results suggest that the antihyperinsulinemic effects of BP are due to the modulation of adipose tissue hypertrophy and adipocytokine secretion. BCEx inhibited the accumulation of visceral fat and serum TG. The study showed that BP and BCEx improve glucose and lipid metabolism disorders caused by a high-fat diet., (© 2013 Asian Oceanian Association for the Study of Obesity . Published by Elsevier Ltd. All rights reserved.)

To examine the presence of CD34-positive cells and intranuclear factors in acute coronary thrombi, we compared thrombi in patients with type 2 diabetes mellitus (DM, n = 21) and without DM (n = 29). Immunohistochemical staining revealed the constitutive presence of platelets, fibrin, erythrocytes, neutrophils, extracellular high-mobility group box 1 (HMGB-1), and histone H3 in all thrombi. There were significantly more oval or flat CD34-positive cells and significantly larger HMGB-1-positive areas in the thrombi from patients with DM. The flat CD34-positive cells expressed ecto-nucleoside triphosphate diphosphohydrolase (a platelet aggregation inhibitor). The number of CD34-positive cells was negatively correlated with the serum levels of glucose and hemoglobin A1c, whereas the HMGB-1-positive area was positively correlated with the levels of serum glucose. The paucity of CD34-positive cells and the high levels of HMGB-1 expression in acute coronary thrombi from patients with type 2 DM could facilitate thrombus formation., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Objective: High-mobility group box 1 (HMGB1) is a damage-associated molecular pattern molecule, which suggests a potential role of this protein in the pathophysiology of acute coronary syndrome (ACS). Circulating HMGB1 has been shown to be independently associated with cardiac mortality in ST-segment elevation myocardial infarction. However, its prognostic value remains unclear in unstable angina and non-ST-segment elevation myocardial infarction (UA/NSTEMI)., Methods: HMGB1, high-sensitivity C-reactive protein (hsCRP), cardiac troponin I and B-type natriuretic peptide concentrations were measured on admission in 258 consecutive patients (mean age of 67 years) hospitalized for UA/NSTEMI within 24h (mean, 7.4h) of the onset of chest symptoms., Results: A total of 38 (14.7%) cardiovascular deaths, including 10 in-hospital deaths, occurred during a median follow-up period of 49 months after admission. In a stepwise Cox regression analysis including 19 well-known clinical predictors of ACS, HMGB1 [relative risk (RR) 3.24 per 10-fold increment; P = 0.0003], cardiac troponin I (RR 1.83 per 10-fold increment, P = 0.0007), Killip class>1 (RR 4.67, P = 0.0001) and age (RR 1.05 per 1-year increment, P = 0.03), but not hsCRP, were independently associated with cardiovascular mortality. In-hospital and cardiovascular mortality rates were higher in patients with increased HMGB1 (≥ 2.4 ng/mL of median value) than those without increased HMGB1 (6.3% vs. 1.5%, P = 0.04; and 23% vs. 6.9%, P = 0.0003)., Conclusion: Circulating concentration of HMGB1 on admission may be a potential and independent predictor of cardiovascular mortality in patients hospitalized for UA/NSTEMI within 24h of onset., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background and Aims: In the present study, we describe a newly developed microchip-based analytical system to evaluate white thrombus formation (WTF). Efficacies of various antithrombotic agents were compared under different flow conditions., Methods: Whole blood containing corn trypsin inhibitor was perfused over a microchip coated with collagen and tissue thromboplastin at the lower and higher shear rates of 240 and 600 s(-1) , and WTF process inside the microchip was quantified by monitoring a flow pressure. Parameters of T(10) (time to 10 kPa), T(10-80) (time from 10 to 80 kPa) and OT (occlusion time; time to 80 kPa) were used to evaluate the onset and the growth rate of WTF, and the capillary occlusion, respectively., Results: After perfusion was started, white thrombus composed of activated platelets and fibrin was formed on the coated surface. Thrombus gradually increased in size and eventually occluded the capillary. Among anticoagulants, heparin (0.5-1.0 U mL(-1)) potently prolonged T(10) at both shear rates, whereas low molecular weight heparin (1.0-2.0 IU mL(-1)) inhibited the growth of WTF at the lower shear rate. Among antiplatelet agents, abciximab (1-2 μg mL(-1)) significantly reduced the size and number of thrombi, which was additively enhanced in the presence of heparin (0.5 U mL(-1) ). OS-1 (specific GPIbα-antagonist) prevented the complete capillary occlusion., Conclusion: The novel monitoring system of WTF may be useful in preclinical and clinical evaluations of different types of antithrombotic strategies, and their effects in combination., (© 2011 International Society on Thrombosis and Haemostasis.)

Thrombomodulin (TM) is an endothelial anticoagulant cofactor that promotes thrombin-mediated activation of protein C. Recently, we conducted a multicentre, double-blind, randomized trial to evaluate the efficacy and safety of recombinant human soluble thrombomodulin (rhsTM, also known as ART-123) for the treatment of disseminated intravascular coagulation (DIC), and found that rhsTM therapy is more effective and safer than low-dose heparin therapy. Thus, in 2008, rhsTM (Recomodulin) was approved for the treatment of DIC in Japan. Here we re-evaluate the therapeutic basis of this drug from the view of its anticoagulant, anti-inflammatory, and cytoprotective properties. Structurally, the extracellular portion of TM is composed of three domains: an N-terminal lectin-like domain (TM-D1), followed by an epidermal growth factor (EGF)-like domain (TM-D2), and an O-glycosylation-rich domain (TM-D3). TM-D2 and TM-D3 are important for the protein's anticoagulant cofactor activities, i.e. inhibition of thrombin and activation of protein C. TM-D1 plays an important role in attenuation of inflammatory responses, through inhibition of leukocyte adhesion to endothelial cells, inhibition of complement pathways, neutralization of lipopolysaccharide (LPS), and sequestration and degradation of pro-inflammatory high-mobility group box 1 protein (HMGB1). Thus, TM on the surface of endothelial cells prevents dissemination of pro-coagulant and pro-inflammatory molecules, and by doing so, allows these molecules to act locally at the site of injury. In patients with sepsis and DIC, TM expression is down-regulated, which may result in dissemination of pro-coagulant and pro-inflammatory molecules throughout the systemic circulation. Replacement with rhsTM may offer therapeutic value in such conditions., (© 2011 International Society on Thrombosis and Haemostasis.)

Stromal cell-derived factor-1 (SDF-1) causes chemotaxis of CXCR4-expressing bone marrow-derived cells. SDF-1 is involved in the pathogenesis of various vascular diseases, including those of the eye. However, the role of SDF-1 in neuronal diseases is not completely understood. Here, we show higher SDF-1 levels in the vitreous humor of patients with retinal detachment (RD) compared with normal patients. SDF-1 correlated positively with the duration as well as the extent of RD. Furthermore, SDF-1 correlated significantly with levels of interleukin-6 and interleukin-8, but not with vascular endothelial growth factor. Western blot analysis results showed significant SDF-1 up-regulation in detached rat retinas compared with normal animals. Immunohistochemistry data showed that SDF-1 was co-localized with the glial cells of the detached retina. SDF-1 blockade with a neutralizing antibody increased photoreceptor cell loss and macrophage accumulation in the subretinal space. The retinal precursor cell line R28 expressed CXCR4. SDF-1 rescued serum starvation-induced apoptosis in R28 cells and enhanced their ability to participate in wound closure in a scratch assay. Our results indicate a surprising, protective role for SDF-1 in RD. This effect may be mediated directly or indirectly through other cell types.

Proteins are folded properly in the endoplasmic reticulum (ER). Various stress such as hypoxia, ischemia and starvation interfere with the ER function, causing ER stress, which is defined by the accumulation of unfolded protein (UP) in the ER. ER stress is prevented by the UP response (UPR) and ER-associated degradation (ERAD). These signaling pathways are activated by three major ER molecules, ATF6, IRE-1 and PERK. Using HaCaT cells, we investigated ER signaling in human keratinocytes irradiated by environmental doses of ultraviolet B (UVB). The expression of Ero1-L(alpha), an upstream signaling molecule of ER stress, decreased at 1-4h after 10 mJ/cm(2) irradiation, indicating that the environmental dose of UVB-induced ER stress in HaCaT cells, without growth retardation. Furthermore, expression of intact ATF6 was decreased and it was translocated to the nuclei. The expression of XBP-1, a downstream molecule of IRE-1, which is an ER chaperone whose expression is regulated by XBP-1, and UP ubiquitination were induced by 10 mJ/cm(2) UVB at 4h. PERK, which regulates apoptosis, was not phosphorylated. Our results demonstrate that UVB irradiation generates UP in HaCaT cells and that the UPR and ERAD systems are activated to protect cells from UVB-induced ER stress. This is the first report to show ER signaling in UVB-irradiated keratinocytes., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Endocannabinoids including anandamide (AEA) and 2-arachidonoylglycerol (2-AG) are important lipid mediators for immunosuppressive effects and for appropriate homeostasis via their G-protein-coupled cannabinoid (CB) receptors in mammalian organs and tissues, and may be involved in wound healing in some organs. The physiological roles of endocannabinoids in periodontal healing remain unknown. We observed upregulation of the expression of CB1/CB2 receptors localized on fibroblasts and macrophage-like cells in granulation tissue during wound healing in a wound-healing model in rats, as well as an increase in AEA levels in gingival crevicular fluid after periodontal surgery in human patients with periodontitis. In-vitro, the proliferation of human gingival fibroblasts (HGFs) by AEA was significantly attenuated by AM251 and AM630, which are selective antagonists of CB1 and CB2, respectively. CP55940 (CB1/CB2 agonist) induced phosphorylation of the extracellular-regulated kinases (ERK) 1/2, p38 mitogen-activated protein kinase (p38MAPK), and Akt in HGFs. Wound closure by CP55940 in an in-vitro scratch assay was significantly suppressed by inhibitors of MAP kinase kinase (MEK), p38MAPK, and phosphoinositol 3-kinase (PI3-K). These findings suggest that endocannabinoid system may have an important role in periodontal healing., (2010 Elsevier Inc. All rights reserved.)

Aquaporin-4 (AQP4) plays a role in the generation of post-ischemic edema. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the central nervous system (CNS) associated with altered brain water balance. Edaravone, a free radical scavenger, is used for the treatment of acute ischemic stroke (AIS) in Japan. In this study, edaravone significantly reduced the infarct area and improved the neurological deficit scores at 24h after reperfusion in a rat transient focal ischemia model. Furthermore, edaravone markedly reduced AQP4 immunoreactivity and protein levels in the cerebral infarct area. In light of observations that edaravone specifically inhibited AQP4 in a rat transient focal ischemia model, we propose that edaravone might reduce cerebral edema through the inhibition of AQP4 expression following cerebral infarction.

The Japanese apricot, known as Ume in Japanese, has been a traditional Japanese medicine for centuries, and is a familiar and commonly consumed food. The health benefits of Ume are now being widely recognized and have been strengthened by recent studies showing that MK615, an extract of compounds from Ume, has strong anticancer and anti-inflammatory effects. However, the potential role of MK615 in the periodontal field remains unknown. Here, we found that MK615 significantly reduced the production of pro-inflammatory mediators (tumor necrosis factor-alpha and interleukin-6) induced by Porphyromonas gingivalis lipopolysaccharide (LPS), a major etiological agent in localized chronic periodontitis, in murine macrophage-like RAW264.7 cells. MK615 markedly inhibited the phosphorylation of ERK1/2, p38MAPK, and JNK, which is associated with pro-inflammatory mediator release pathways. Moreover, MK615 completely blocked LPS-triggered NF-kappaB activation. The present results suggest that MK615 has potential as a therapeutic agent for treating inflammatory diseases such as periodontitis.

1,5-anhydro-d-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits antioxidant and antibacterial activity, and inhibits cytokine release by attenuating NF-kappaB activation in LPS-stimulated mice. The present study examined whether 1,5-AF inhibits lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) in vitro and in vivo. We found that 1,5-AF significantly blocked the production of NO, and protein and mRNA expression of iNOS, and up-regulated IL-10 production in vitro. We also investigated the effects of 1,5-AF on acute lung inflammation in C57BL/6J mice. We found that protein and mRNA expression of iNOS in lung tissues were inhibited by 1,5-AF pretreatment. In addition, the serum level of IL-10 was upregulated by 1,5-AF. Collectively, the iNOS transcriptional and translational inhibitory effects of 1,5-AF seem to be prolonged and enhanced by the production of IL-10. These results suggest that 1,5-AF could be a useful adjunct in the treatment of acute lung inflammation.

High mobility group box-1 (HMGB1), a non-histone DNA-binding protein, is massively released into the extracellular space from neuronal cells after ischemic insult and exacerbates brain tissue damage in rats. Minocycline is a semisynthetic second-generation tetracycline antibiotic which has recently been shown to be a promising neuroprotective agent. In this study, we found that minocycline inhibited HMGB1 release in oxygen-glucose deprivation (OGD)-treated PC12 cells and triggered the activation of p38mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERK1/2). The ERK kinase (MEK)1/2 inhibitor U-0126 and p38MAPK inhibitor SB203580 blocked HMGB1 release in response to OGD. Furthermore, HMGB1 triggered cell death in a dose-dependent fashion. Minocycline significantly rescued HMGB1-induced cell death in a dose-dependent manner. In light of recent observations as well as the good safety profile of minocycline in humans, we propose that minocycline might play a potent neuroprotective role through the inhibition of HMGB1-induced neuronal cell death in cerebral infarction.

Purpose: To evaluate vitreous vascular endothelial growth factor (VEGF), stromal cell-derived factor 1alpha (SDF-1alpha), interleukins (ILs), and tumor necrosis factor-alpha (TNF-alpha) after intravitreal bevacizumab or triamcinolone acetonide (TA) in eyes with proliferative diabetic retinopathy (PDR)., Design: Interventional, consecutive, retrospective, comparative study with a historical control., Participants: Forty-seven eyes of 47 patients affected by active PDR were investigated. Bevacizumab (1.25 mg; 19 eyes; bevacizumab group) or TA (4 mg; 10 eyes; TA group) was injected into the vitreous cavity as preoperative adjunctive therapy 7 days before vitrectomy. Eighteen eyes without injection served as controls (control group)., Methods: The vitreous samples were obtained at vitrectomy and were analyzed for concentrations of total protein, VEGF, SDF-1alpha, IL-1beta, IL-6, IL-8, IL-10, IL-12p70, and TNF-alpha., Main Outcome Measures: Vitreous concentrations of VEGF, SDF-1alpha, ILs, and TNF-alpha were compared among bevacizumab, TA, and control groups., Results: Vitreous concentrations of VEGF and SDF-1alpha were lower in bevacizumab and TA groups compared with the control group. The median VEGF level was 0 pg/ml (range, 0-79.2 pg/ml) in the bevacizumab group, 343.5 pg/ml (range, 0-1683.3 pg/ml) in the TA group, and 1202.5 pg/ml (range, 76-4213.9 pg/ml) in the control group. The median SDF-1alpha level was 149.2 pg/ml (range, 0-519.4 pg/ml) in the bevacizumab group, 87.5 pg/ml (range, 0-252.5 pg/ml) in the TA group, and 245.7 pg/ml (range, 0-856.8 pg/ml) in the control group. The differences in both vitreous VEGF and SDF-1alpha concentrations among 3 groups were statistically significant (P<0.001 and P = 0.010, respectively). The eyes with intravitreal bevacizumab demonstrated the lowest vitreous level of VEGF, and the level was statistically significant compared with the eyes with intravitreal TA and control eyes (P<0.001 and P<0.001, respectively). The control eyes had the highest vitreous level of SDF-1alpha, and the level was statistically significant compared with the eyes with intravitreal bevacizumab and TA (P = 0.015 and P = 0.009, respectively). There was no significant difference regarding ILs and TNF-alpha., Conclusions: Intravitreal injection of bevacizumab potentially diminishes not only VEGF but also SDF-1alpha. These findings suggest that intravitreal bevacizumab may influence intraocular mediators beyond VEGF., Financial Disclosure(s): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Lipopolysaccharide (LPS) stimulates macrophages by activating NF-kappaB, which contributes to the release of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6. 1,5-Anhydro-D-fructose (1,5-AF), a monosaccharide formed from starch and glycogen, exhibits anti-oxidant activity and enhances insulin secretion. This study examined the effects of 1,5-AF on LPS-induced inflammatory reactions and elucidated its molecular mechanisms. Before LPS challenge, mice were pretreated with 1,5-AF (38.5 mg/kg). We found that 1,5-AF pretreatment attenuated cytokine release into the serum, including TNF-alpha, IL-6 and macrophage chemoattractant protein (MCP)-1. Furthermore, pretreatment with 1,5-AF (500 microg/ml) attenuated cytokine release, and 1,5-AF directly inhibited the nuclear translocalization of the NF-kappaB p65 subunit in LPS-stimulated murine macrophage-like RAW264.7 cells. This inhibition was responsible for decreased LPS-induced phosphorylation on Ser536 of the NF-kappaB p65 subunit, which is a posttranslational modification involved in the non-canonical pathway. Collectively, these findings indicate that the anti-inflammatory activity of 1,5-AF occurs via inactivation of NF-kappaB.

Thrombomodulin (TM) is an endothelial cell surface anticoagulant glycoprotein that performs antimetastatic, angiogenic, adhesive, and anti-inflammatory functions in various tissues. It is also expressed in epidermal keratinocytes. We found that a physiological dose (10mJ/cm(2)) of mid-wavelength ultraviolet irradiation (UVB) significantly induced TM expression via the p38mitogen-activated protein kinase (MAPK)/cyclic AMP response element (CRE) signaling pathway in the epidermal keratinocyte cell line HaCaT; this shows that TM regulates the survival of HaCaT cells. SB203580, a p38MAPK inhibitor, significantly decreased TM expression and the viability of cells exposed to UVB. Furthermore, overexpression of TM markedly increased cell viability, and it was abrogated by TM small interfering RNA (siRNA), suggesting that TM may play an important role in exerting cytoprotective effect on epidermal keratinocytes against low-dose UVB.

Vascular endothelial growth factor (VEGF) has been noted in the pathogenesis of atherosclerosis. To examine the usefulness of the serum concentration of VEGF as an index of atherosclerosis, we analyzed the serum VEGF concentrations in 443 adults who underwent a medical checkup. The mean serum VEGF concentration of men (229+/-147 pg/ml) was significantly higher than that of women (182+/-112 pg/ml). The platelet count showed a slight correlation with the serum VEGF concentration in both genders (men R=0.287, women R=0.296), corresponding with the results of experiments that platelets are the major source of VEGF in circulating peripheral blood. In men, the serum VEGF concentrations correlated with platelet counts, body fat percentages, leukocyte counts, and HDL-cholesterol concentrations (negative correlation). In the multiple regression analysis performed for men's serum VEGF concentrations, the decision coefficient (R2) was maximized (R2=0.173) when the leukocyte count, the body fat percentage, and the HDL-cholesterol concentration were taken into account besides the platelet count. Male smokers' serum VEGF concentrations were higher than non-smokers'. Smoking in men significantly affected the sex difference in the serum VEGF concentration, leukocyte count, and HDL-cholesterol concentration. We concluded that the serum VEGF concentration might be closely related to atherosclerosis accelerating factor, especially in men.

The developmental timing of all types of cells must be synchronized and spatially coordinated to achieve the organized development of a multicellular organism. Previously, we found RNAi of asb-1, encoding a germline-specific isoform of mitochondrial ATP synthase b subunit, caused 100% penetrant sterility in Caenorhabditis elegans. ATP synthase is one of the five complexes of the mitochondrial respiratory chain, and defects in some of the components of the chain are known to slow the growth and extend the lifespan of worms. We found that development of asb-1 mutant germ line was not arrested at any stage, but did slow to half the rate of wild type, whereas the rate of somatic development was the same in asb-1 mutants as that of wild type, indicating that asb-1 is required to maintain the rate of germline development but has no effect on somatic development. Among ATP synthase subunit genes, RNAi of asg-1, encoding a germline-specific isoform of the g subunit, also caused asb-1-like sterility, indicating that some other germline-specific components are also required to maintain the rate of germline development. Both asb-1 and asg-1 are located on autosomes while they possess counterparts, asb-2 and asg-2, respectively, on X chromosome, which are both required for somatic development. Chromosomal locations of the genes may be the basis of the segregation of germline/somatic functions of each gene, as were demonstrated for other autosomal/X-linked duplicated gene pairs.

We have been examining the role of heat shock factor 1 (HSF1) in the pleiotropic effects of statins. In parallel studies, we found that statin induces the nuclear translocation of HSF1 and that a decoy oligonucleotide encoding the heat shock element inhibits the statin-induced expression of heat shock protein 70, endothelial nitric oxide synthase (eNOS) and thrombomodulin. Also, in vascular endothelial cells, increases in the expression of human HSF1 corresponded with elevated steady-state levels of eNOS and thrombomodulin and reduced levels of endothelin-1 and plasminogen activator inhibitor-1. We also found that heat shock proteins induced eNOS and thrombomodulin expression and reduced PAI-1 and ET-1 expression. In particular, a combination of HSP70 and HSP90 strongly induced eNOS expression and reduced PAI-1 expression. In the current studies, we generated a constitutively active form of HSF1 and found that it is more effective than the wild-type HSF at inducing thrombomodulin and eNOS expression and decreasing endothelin-1 and plasminogen activator inhibitor-1 expression. These results show that the wild-type and constitutively active forms of HSF1 induce anticoagulation and relaxation factors in vascular endothelial cells and could therefore be used to treat cardiovascular disease.

Background: Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC., Objectives: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system., Methods: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro., Results: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes., Conclusions: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

High mobility group box 1 (HMGB1) exhibits unique biochemical functions as a biologically intrinsic requisite factor and as a toxin. As such, it is imperative to understand the mechanism by which these seemingly and diametrically opposed functions are exerted. To effectively discriminate these actions is important to accurately and precisely determine the concentration of HMGB1 in biological samples. Research in this fascinating field, however, has been lacking due to the absence of a simple analytical system for HMGB1 that can be adapted for large sample numbers. In this report, we review the physiological and pathological significance of HMGB1 and describe the development of an assay method for this pleiotropic protein.

Background: Soluble thrombomodulin is a promising therapeutic natural anticoagulant that is comparable to antithrombin, tissue factor pathway inhibitor and activated protein C., Objectives: We conducted a multicenter, double-blind, randomized, parallel-group trial to compare the efficacy and safety of recombinant human soluble thrombomodulin (ART-123) to those of low-dose heparin for the treatment of disseminated intravascular coagulation (DIC) associated with hematologic malignancy or infection., Methods: DIC patients (n = 234) were assigned to receive ART-123 (0.06 mg kg(-1) for 30 min, once daily) or heparin sodium (8 U kg(-1) h(-1) for 24 h) for 6 days, using a double-dummy method. The primary efficacy endpoint was DIC resolution rate. The secondary endpoints included clinical course of bleeding symptoms and mortality rate at 28 days., Results: DIC was resolved in 66.1% of the ART-123 group, as compared with 49.9% of the heparin group [difference 16.2%; 95% confidence interval (CI) 3.3-29.1]. Patients in the ART-123 group also showed more marked improvement in clinical course of bleeding symptoms (P = 0.0271). The incidence of bleeding-related adverse events up to 7 days after the start of infusion was lower in the ART-123 group than in the heparin group (43.1% vs. 56.5%, P = 0.0487)., Conclusions: When compared with heparin therapy, ART-123 therapy more significantly improves DIC and alleviates bleeding symptoms in DIC patients.

Statins not only reduce serum cholesterol but they also improve vascular endothelial function independent of their lipid-lowering effects. However, except for the mechanism of nitric oxide induction via calveolin, the physiologic basis for the pleiotropic effect of statins remains unknown. In the present study, we investigated the relationship between the effects of statins on vascular endothelial cell function and heat shock proteins. We found that, in vascular endothelial cells, simvastatin increased the steady-state levels of heat shock proteins 90 and 70, and heme oxygenase-1 and caused the nuclear translocation of heat shock factor 1. A decoy oligonucleotide encoding the heat shock element inhibited statin-induced expression of heat shock protein 70, endothelial nitric oxide synthase, and thrombomodulin. This decoy oligonucleotide also inhibited the ability of statin to reduce endothelin-1 and plasminogen activator inhibitor-1 expression. These results indicate that statins improve vascular endothelial function via heat shock factor 1, which may contribute to their ability to improve cardiovascular disease.

Background: High-sensitivity sandwich ELISA methods have been developed using chemiluminescent substrates. HMGB1 (high mobility group box 1) protein has been shown to play a critical role in several inflammatory diseases and it may be involved in the development of atherosclerosis., Methods: Anti-human HMGB1 monoclonal antibodies and anti-peptide polyclonal antibodies against the peptide sequence (KPDAAKKGVVKAEK) with high antigenicity and different from the sequence of HMGB2 were developed, and the antibodies were used to construct sandwich ELISA methods with a chromogenic substrate (TMBZ) and a chemiluminescent substrate (PS-atto). Highly purified human HMGB1 was used as a standard material and high-sensitivity CRP was measured to compare with HMGB1., Results: The analytical characteristics of the ELISA method we developed were validated inter-assay and intra-assay CVs were <10%, and the detection limit was 0.3 microg/l by the chemiluminescent method and 1 microg/l with the chromogenic substrates. HMGB1 was detected in the serum of patients with acute coronary syndrome (ACS). When a cut-off of 0.6 microg/l HMGB1 upon admission to the intensive care unit (ICU) was used, the risk of developing an acute cardiac event within 1 month after discharge of ACS patients with an abnormal HMGB1 was significantly higher than for the patients with normal values (P<0.0001). The usefulness of HMGB1 as an acute prognostic marker was suggested., Conclusions: The assay is easy to perform and suitable for use in the hospital laboratory and for screening large populations. HMGB1 is detectable in the serum of ACS patients and that the serum concentration of HMGB1 may be a prognostic indicator in ACS patients.

Background: In 1997, Stoppini et al reported that monoclonal antibody specific to the C-terminal 92-99 of beta(2)-microglobulin (beta(2)m) had been capable of inhibiting fibrillogenesis of beta(2)m in vitro. Meanwhile, recent studies have indicated that an acidifying procedure can unfold conformation of the precursor protein, leading to fibril formation of beta(2)m as well as a transthyretin., Methods: We thus prepared monoclonal antibody specific to the C-terminal 92-99 (mAb 92-99), and investigated its reactivity in plasma ultrafiltrate and amyloid tissues from 18 hemodialysis patients with dialysis-related amyloidosis (DRA)., Results: beta(2)m extracted from ultrafiltrate showed no reaction for mAb 92-99, whereas acidified beta(2)m from ultrafiltrate showed a reaction for mAb 92-99. Similarly, a homogenate of carpal amyloid tissues showed a strong reaction for mAb 92-99 on immunoblotting. Immunohistochemical study showed also a distinct staining for mAb 92-99 in 7 Congophilic specimens from DRA patients. More interestingly, staining for mAb 92-99 could be found in most, though not all, non-Congophilic tissues., Conclusion: This study demonstrates that the monoclonal antibody specific to the C-terminal 92-99 of beta(2)m can detect the conformational intermediate in amyloidogenesis of beta(2)m ex vivo, and demonstrates that an unfolded beta(2)m at C-terminal could be found not only in Congophilic area but even in non-Congophilic area as well.

Background: The presence of alpha2-macroglobulin (alpha2M) in amyloid tissue from patients with dialysis-related amyloidosis (DRA) was demonstrated by Argilés et al in 1989. Thereafter, the formation of the complex of beta2-microglobulin (beta2m) with alpha2m was confirmed directly by in vitro study. In Alzheimer's disease, complex formation of amyloid beta-peptide and alpha2M is considered to play an important role in the pathogenesis by modifying the degradation processes of amyloid protein. Thus, we hypothesized that the alpha2M-beta2m complex is an important factor in the pathogenesis of DRA as well. Here, we measured the circulating levels of alpha2M-beta2m complex in the maintenance hemodialysis patients and discussed about its clinical significance in DRA., Methods: One hundred and thirty-seven hemodialysis patients and 11 prehemodialysis chronic renal failure (CRF) patients were included in this study. The affinity of purified alpha2M for beta2m was confirmed by a highly sensitive 27 MHz quartz crystal microbalance (QCM). The presence of circulating alpha2M-beta2m complex was analyzed by immunoblotting analysis. Furthermore, the serum levels of alpha2M-beta2m complex were measured by sandwich enzyme immunoassay., Results: QCM analysis revealed the high affinity of alpha2M for beta2m. The presence of circulating alpha2M-beta2m complex was detected in two out of a total 11 prehemodialysis CRF patients and in 95 out of the total of 137 hemodialysis patients. None of the healthy subjects, however, were observed to present with any alpha2M-beta2m complex. Serum levels of the alpha2M-beta2m complex were correlated to the duration of hemodialysis (P= 0.043). Serum levels of the alpha2M-beta2m complex were significantly higher in patients with high DRA score than in patients with negative DRA score (P= 0.018). Moreover, serum levels of the alpha2M-beta2m complex showed significantly lower in the hemodiafiltration patients compared to the hemodialysis patients (P= 0.002) and showed a strong correlation with DRA score in hemodialysis patients excluding 11 hemodiafiltration patients (P= 0.0004)., Conclusion: This study is the first to demonstrate the presence of circulating alpha2M-beta2m complex in hemodialysis patients. Furthermore, we observed the correlation between serum levels of alpha2M-beta2m complex and clinical characteristics of DRA. Thus we concluded that a formation of an alpha2M-beta2m complex may be implicated in DRA.

Background: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm., Objectives: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis., Methods: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed., Results and Conclusions: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-alpha, and TGF-beta.

In the present study, we generated killer cells specific for hepatitis C virus (HCV) structural protein by re-stimulation of immune spleen cells from H-2(d) haplotype transgenic (Tg) mice, expressing the core, E1, E2, and NS2 genes of HCV regulated by the Cre/loxP switching system. The generated killer cells were conventional CD8(+)L(d) class-I MHC molecule-restricted cytotoxic T lymphocytes (CTLs) and specific for the HCV E1 structural protein. Because the CTLs could also kill hepatocytes from the Tg mice expressing HCV structural proteins in vitro, we attempted to transfer those CTLs intravenously into interferon regulatory factor-1 (IRF-1) negative, CD8-deficient Tg mice representing the HCV structural genes on hepatocytes to examine whether the inoculated CD8(+) CTLs can eliminate hepatocytes expressing the HCV genes in vivo. We observed an elevation of serum ALT level as well as damage of the liver tissue histologically. To our knowledge, this is the first demonstration to show that HCV-specific CD8(+) CTLs specifically attack hepatocytes expressing the HCV structural proteins both in vitro and in vivo.

Cancer-associated retinopathy (CAR) is an ocular manifestation of a paraneoplastic syndrome whereby immunological reactions toward recoverin (Rec), a retina-specific Ca(2+) binding protein, and its aberrant expression in tumor cells lead to the retinal degeneration. To elucidate functional roles of the aberrantly expression in cancer cells, we performed immunoprecipitation using anti-human Rec mAb. We observed co-precipitation of G-protein-coupled receptor kinases (GRKs) and caveolin-1 with Rec from cell lysates of 293 or SSTW cells. Immunocytochemistry revealed that immunoreactivities toward Rec within the cancer cells were almost identical to those toward GRKs and caveolin-1. The present data strongly suggest that aberrantly expressed Rec should be involved in the GRK-dependent cellular regulation in cancer cells.

The epidermal growth factor receptor plays crucial roles throughout the development of multicellular organisms, and inappropriate activation of the receptor is associated with neoplastic transformation of many cell types. The receptor is thought to be activated by ligand-induced homodimerisation. Here, however, we show by chemical cross-linking and sucrose density-gradient centrifugation that in the absence of bound ligand the receptor has an ability to form a dimer and exists as a preformed dimer on the cell surface. We also analysed the receptor dimerisation by inserting cysteine residues at strategic positions about the putative alpha-helix axis of the extracellular juxtamembrane region. The mutant receptors spontaneously formed disulphide bridges and transformed NIH3T3 cells in the absence of ligand, depending upon the positions of the cysteine residue inserted. Kinetic analyses of the disulphide bonding indicate that EGF binding induces flexible rotation or twist of the juxtamembrane region of the receptor in the plane parallel with the lipid bilayer. The binding of an ATP competitor to the intracellular domain also induced similar flexible rotation of the juxtamembrane region. All the disulphide-bonded dimers had flexible ligand-binding domains with the same biphasic affinities for EGF as the wild-type. These results demonstrate that ligand binding to the flexible extracellular domains of the receptor dimer induce rotation or twist of the juxtamembrane regions, hence the transmembrane domains, and dissociate the dimeric, inactive form of the intracellular domains. The flexible rotation of the intracellular domains may be necessary for the intrinsic catalytic kinase to become accessible to the multiple tyrosine residues present in the regulatory domain and various substrates, and may be a common property of many cell-surface receptors, such as the insulin receptor., (Copyright 2001 Academic Press.)

Anandamide (ANA) and 2-arachidonoylglycerol (2-AG), two endogenous cannabinoids, can be generated by activated macrophages and platelets, respectively, in the context of endotoxic shock, and are proposed to play a crucial role in the induction of the shock-related hypotension. Taking advantage of our recently discovered function of polymyxin B (PMB) binding to ANA and 2-AG, we developed a new method for measuring ANA and 2-AG by applying PMB-immobilized beads to selectively adsorb them in biological fluids, instead of organic solvent extraction. The eluate from beads can be directly fractionated by reverse-phase high-performance liquid chromatography (HPLC), and the fractionations corresponding to authentic ANA and 2-AG are collected and derivatized with fluorogenic reagent and subsequently quantified by HPLC with fluorometric detection. The calibration graphs of ANA and 2-AG were linear over a range of 1 to 500 pmol/ml. The limits of detection for ANA and 2-AG were 20 and 50 fmol, respectively. Intraassay precision was 2.24-4.25 and 3.47-5.44%, and interassay was 4.05-6.14 and 4.92-7.28% for ANA and 2-AG, respectively. Using this method, we first determined a 4-fold and 3-fold higher level of ANA and 2-AG, respectively, in the sera of patients with endotoxic shock than in normal serum. This finding should help in elucidating the role of the endogenous cannabinoids in the hypotension of human endotoxic shock. This method is rapid, sensitive, and reliable for simultaneously quantifying ANA and 2-AG in biological fluids, and has potential for clinical usage., (Copyright 2001 Academic Press.)

The pathogenesis of tuberculous meningitis is still unclear. Recently, vascular endothelial growth factor (VEGF) was found to be associated with inflammatory diseases and we found the increased serum level of VEGF in pulmonary tuberculosis. We hypothesized that VEGF might be associated with the pathogenesis of tuberculous meningitis and measured serum and cerebrospinal fluid (CSF) levels of VEGF in 28 patients with tuberculous meningitis and 31 non-tuberculous infectious meningitis patients (13 bacterial meningitis patients, eight fungal meningitis patients and 10 patients with viral meningitis) before therapy. We examined the CSF VEGF levels 3 months after in 12 tuberculous meningitis patients. The serum and CSF levels of VEGF were significantly higher in tuberculous meningitis than in other meningitis. The decrease in titer of CSF VEGF paralleled the clinical improvement of tuberculous meningitis. Immunohistochemical staining of autopsied brains demonstrated the presence of VEGF in the inflammatory mononuclear cells of the dense fibroconnective tissue both in the subarachnoid space and surrounding the vasculitis lesion. We found the expression of VEGF in tuberculous meningitis and think that VEGF reflects its activity.

Interleukin-8 (IL-8) mRNA was constitutively expressed in human hepatoma cell line, HepG2 and in human hepatocellular carcinoma (HCC), which often form hypervascular tumors. The sequence 5'-AGGAAG-3' at -137 to -132 bp of IL-8 promoter was shown to be polyomavirus enhancer A binding protein-3 (PEA3) binding site, which can cooperate with activator protein-1 (AP-1). Both PEA3 and AP-1 are essential for constitutive IL-8 expression in HepG2 cells, determined by promoter assays. Moreover, PEA3 and IL-8 proteins coexisted in HCC tissues, but not in uninvolved liver tissues. It is possible PEA3 may have important roles in tumor progression and in angiogenesis in HCC., (Copyright 2000 Academic Press.)

Recent studies have implicated acetylation of several nuclear proteins such as histones and p53 on their epsilon-portion of lysine residues in eukaryotic transcription. Here we raised a specific polyclonal antibody against epsilon-acetylated lysine. Using the antibody, we detected hypernuclear acetylation (HNA) in atherosclerotic vascular smooth muscle cells (VSMCs). Thrombin, a humoral factor known to cause activation and proliferation of VSMCs, strongly potentiated HNA in cultured VSMCs. MAP kinase pathway and a signal coactivator CREB binding protein (CBP) were involved in thrombin-induced HNA of VSMCs. Our results suggest that coactivators cooperating with signal-dependent transcription activators play an important role in atherosclerogenesis via HNA in VSMCs., (Copyright 1999 Academic Press.)

Mapping of protein domains having a distinct function is essential to understanding the protein's structure-function relationship. We used a bacteriophage lambda surface expression vector, lambdafoo, in order to determine the minimal carbohydrate-binding domain of human galectin-3 (Gal-3). Gal-3 cDNA was randomly digested by DNase I and cloned into the phage vector. The library generated was screened by affinity selection using lactose immobilized on agarose beads. DNA sequence analysis of a set of isolated clones defined the minimal folding domain of Gal-3 required for lactose binding, which consisted of 136 amino-acid residues. Using the phage clones isolated, we also determined relative dissociation constants in solution between lactose and the minimal domain expressed on the phage surface. This technique does not require either purified or labeled proteins, and bacteriophage lambda surface display may, therefore, be useful for protein domain mapping and in vitro studies of various macromolecular interactions., (Copyright 1999 Academic Press.)

Haemophilia A patients who receive repeated transfusion of fVIII concentrates often develop inhibitor alloantibodies, resulting in reduced efficacy of the therapy. Determination of fVIII epitopes for the alloantibodies is essential for an understanding of their inhibitory effect on blood coagulation. Random fragments of fVIII displayed on lambda phage particles were selected using two patient plasmas immobilized onto the surface of a microtiter plate. A set of clones defined the minimal domain that consisted of 157 amino acid residues including cysteine at both boundaries. The minimal domain absorbed most of the binding activities of the plasmas to fVIII, suggesting that the domain contains a major determinant for the plasmas. Site-directed mutagenesis and chemical denaturation of the domain confirmed that a tertiary structure formed by the disulfide bridge was recognized by the antibodies. The epitope domain defined overlaps with fVIII binding sites to vWf and phospholipid, and may play an important role in blood coagulation. Thus, the bacteriophage lambda surface display may be useful for mapping the minimal folding domain of various protein antigens that contain a conformational epitope.

The aspartate chemoreceptor Tar of Escherichia coli serves as a warm sensor that produces attractant and repellent signals upon increases and decreases in temperature, respectively. However, increased levels of methylation of the cytoplasmic domain of Tar resulting from aspartate binding convert Tar to a cold sensor with the opposite signaling behavior. Detailed analyses of the methylation sites, which are located in two separate alpha-helices (MH1 and MH2), have suggested that intra- and/or intersubunit interactions of MH1 and MH2 play a critical role in thermosensing. These interactions may be influenced by binding of aspartate, which could trigger some displacement of MH1 through the second transmembrane region (TM2). As an initial step toward understanding the role of TM2 in thermosensing, we have examined the thermosensing properties of 43 mutant Tar receptors with randomized TM2 sequences (residues 190-210). Among them, we identified one mutant receptor (Tar-I2) that functioned as a cold sensor in the absence of aspartate. This is the first example of attractant-independent inversion of thermosensing in Tar. Further analyses identified the minimal essential divergence from the wild-type Tar sequence (Q191V-W192R-Q193C) required for the inverted response. Thus, displacements of TM2 seem to influence the thermosensing function of Tar., (Copyright 1999 Academic Press.)

We devised a simple method to maintain an immortalized newborn rat keratinocyte cell line at the air-liquid interface using a tissue culture insert fitted with a microporous membrane. The cells formed stratified layers of flattened and anucleated cells resembling stratum corneum of the epidermis. Deiminated proteins, which are localized in the cornified layer of epidermis as the reaction products of peptidylarginine deiminase were detected immunohistochemically in the differentiated cells. Western blot analyses revealed that major deiminated proteins were type I keratins K10 and K14. Deiminated products of type II keratin K5 were found as minor components. Our observations show that deimination of keratins might be correlated with terminal differentiation of the immortalized keratinocyte cell line.

We examined the induction of inflammatory cytokines (including interleukin-1, interleukin-6, interleukin-8, and tumor necrosis factor-alpha) by several species of possible causative bacteria in periapical periodontitis. Assays were done on human whole blood cultures from patients with differing numbers of periapical lesions; those having radiographically clear periapical lesions in 10 or more teeth (high lesion group), in one or two teeth (low lesion group), and healthy volunteers having no periapical lesions (no lesion group). Prevotella melaninogenica ATCC 25845 induced interleukin-6 more strongly in subjects from the high lesion group than in the other groups. To ascertain the degree of sensitization by test bacteria, we examined the reactivities of antibodies in serum and saliva from the six subjects to different bacterial species. Porphylomonas gingivalis cells reacted strongly with sera from the high lesion group. Thus, Prevotella melaninogenica and Porphylomonas gingivalis may be involved in multilesional periapical periodontitis by inducing specific cytokines and/or humoral immune responses.

Background: Recent studies demonstrated not only that advanced glycation end product could be found in amyloid tissue from patient with dialysis related amyloidosis, but also that amyloid beta2-microglobulin was modified with N(epsilon)-(carboxymethyl)lysine (CML). We wanted to determine if CML could be a biomarker in these patients., Methods: To raise polyclonal anti-carboxymethyllysine antibody, human serum albumin was carboxymethylated by glyoxylic acid and was immunized to rabbits as antigen. Carboxymethyllysine-hemoglobin (CML-Hb) levels were measured by the dot blotting method using this antibody., Results: The levels of CML-Hb were 6.68 +/- 3.10 nmol CML/mg Hb in nondiabetic hemodialysis patients (N = 70), 6.39 +/- 3.43 nmol CML/mg Hb in diabetic hemodialysis patient (N = 21), and 3.13 +/- 0.88 nmol CML/mg Hb in 47 healthy volunteers. For clinical signs of dialysis-related amyloidosis, 70 nondiabetic hemodialysis patients were scored according Gejyo's criteria. The CML-Hb levels in patients with a high amyloid score as well as a low amyloid score were significantly higher than in patients with negative amyloid score (8.89 +/- 3.53 nmol CMLmg Hb, 7.28 +/- 2.32 nmol CML/mg Hb vs. 5.11 +/- 2.09 nmol CML/mg Hb, P < 0.001, P < 0.05). Furthermore, the CML-Hb levels correlated significantly with serum values of the methylguanidine over creatinine ratio and hyaluronate., Conclusions: We suggest that CML-Hb is increased in blood from patients on maintenance hemodialysis and is thus a potential biomarker of oxidative damage in these patients. Moreover, CML-modification of protein may play a pathogenic role in the development of dialysis related amyloidosis.

Pancreatic beta-cells possess nitric oxide (NO) synthases (NOSs) which synthesize NO and L-citrulline from L-arginine. The present study was designed to explore the mechanism of citrulline and arginine metabolism in beta-cells and its possible coupling to beta-cell functions. The enzymes involved in citrulline-arginine metabolism, argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and NOS were expressed in rat islets and insulinoma HIT T15 cells. In the presence of stimulatory glucose, L-citrulline and L-argininosuccinate at physiological concentrations (0.1-1 mM) increased cytosolic Ca2+ concentration ([Ca2+]i) in rat beta-cells. The citrulline-induced [Ca2+]i increase was inhibited by a NOS inhibitor, N(G)-monomethyl-L-arginine (NMMA). L-citrulline also stimulated NO production in HIT cells, which was inhibited by NMMA. In conclusion, L-citrulline is metabolized by ASS-ASL-NOS cycle to produce NO, which in turn increases [Ca2+]i in beta-cells.

The physiological and pharmacobiological changes associated with space flight are of greater concern. Exposure to a weightless environment has been shown to have numerous effects on body composition and organ functions. Alterations include decreases in muscle and liver mass, changes in bone structure and integrity, and changes in cardiovascular functions. Zero-gravity in particular has been reported to inhibit several physiological processes of bone formation, retard bone growth and impair the mechanical properties of bones. This report examines the effect of 14 days of spaceflight on the bone trace element compositions of rapidly growing rats. Marked changes of bone trace element contents were found in either weight-bearing bones or non-weight-bearing bones, depending on the metal species. Histological examination revealed an irregular thickening of the endosteal surface of the cortical bone (thoracic vertebrae) of the in-flight rat, whereas it was uniform in the ground control. We suggest that the microgravity environment causes several bone alternations, such as abnormal trace element compositions and defects in vertebral maturation.

We have developed plasmid and phage vectors for the display of foreign proteins on the surface of bacteriophage lambda capsid by modifying the D gene which encodes the major head protein gpD. The vectors have multiple cloning sites, and permit colour selection and conditional chain termination for recombinants. Displayed proteins can be fused to either the N or C terminus of gpD by a peptide linker. The conditional chain termination scheme, via a host Escherichia coli suppressor activity, allows the fusion and assembly of homomultimeric proteins as well as control of the number of fusion proteins per phage particle. We have successfully displayed beta-lactamase, IgG-binding domains of the Staphylococcus aureus protein A, and beta-galactosidase by cloning the genes into the vector. The constructs express functionally active proteins fused to gpD that assemble into phage particles. These results suggest that gpD may be fused to many other peptides and proteins at their N or C terminus and the fusion products may be accessible on the surface of bacteriophage lambda particles.